Project Abstract Our understanding of fungal mechanisms that enable success within mammalian hosts is limited. In contrast, multiple virulence systems have been defined for bacterial pathogens, including so-called ?quorum sensing? systems that allow for detection of accumulation self-produced signaling molecules. Such autoregulatory systems permit individual bacterial cells to detect the density of same-species neighbors. Qsp1 is an 11 amino acid peptide of unknown function secreted by C. neoformans. We identified QSP1 as a direct target of three different transcription factors, each of which is required for C. neoformans virulence. Our investigations of Qsp1 function revealed that this peptide is required for virulence, including proliferation of the pathogen in infected lungs and within infected macrophages. In vitro, qsp1? mutants display a density-dependent defect in cell wall integrity and changes in the activities of multiple secreted peptidases. Our studies of Qsp1 biogenesis indicate that it is secreted as precursor that is matured to its final form extracellularly by a cell-associated peptidase. Qsp1-mediated signaling requires a predicted oligopeptide importer in the responding cell, raising the intriguing hypothesis that peptide import is an integral feature of the signaling mechanism, a mechanism that would be without precedent in eukaryotes. Strongly supporting this model, intracellular expression of the mature form of Qsp1 complements multiple phenotypes of a deletion of QSP1. In this application, we propose work that aims to understand how biologically active Qsp1 is produced, determine how cells respond to Qsp1 and understand how this system promotes virulence. Specifically, we will test the hypothesis, supported by preliminary work, that deglycosylation of the peptide is required to activate it after secretion, a mechanism without precedent. In addition, because our data suggest an intracellular site of action, we will elucidate this underlying molecular mechanism by identifying the intracellular receptor(s) for Qsp1, using a combination of candidate and unbiased approaches. Finally, based our preliminary RNA-seq and functional studies, we will test the hypothesis that Qsp1 signaling triggers both the production of toxic extracellular factors and an intracellular program that is self-protective. Through the use of mutants, we will test the model that this program mediates the role of Qsp1 signaling in virulence. Anticipated Impact: 1) the identification of a novel biogenesis mechanism for a signaling peptide involving peptide deglycosylation, 2) the identification of an intracellular receptor for an exported peptide required for virulence, and 3) the identification of Qsp1-regulated extracellular toxic products that promote the virulence of a human fungal pathogen.